Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	12.50-800 pg/mL
Sensitivity:	7.50 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human MCSF in samples. No significant cross-reactivity or interference between Human MCSF and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human MCSF. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human MCSF and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human MCSF, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human MCSF. The concentration of Human MCSF in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	M-CSF: Cytokine that plays an essential role in the regulation of survival, proliferation and differentiation of hematopoietic precursor cells, especially mononuclear phagocytes, such as macrophages and monocytes. Promotes the release of proinflammatory chemokines, and thereby plays an important role in innate immunity and in inflammatory processes. Plays an important role in the regulation of osteoclast proliferation and differentiation, the regulation of bone resorption, and is required for normal bone development. Required for normal male and female fertility. Promotes reorganization of the actin cytoskeleton, regulates formation of membrane ruffles, cell adhesion and cell migration. Plays a role in lipoprotein clearance. Aberrant expression of CSF1 or CSF1R can promote cancer cell proliferation, invasion and formation of metastases. Overexpression of CSF1 or CSF1R is observed in a significant percentage of breast, ovarian, prostate, and endometrial cancers. Aberrant expression of CSF1 or CSF1R may play a role in inflammatory diseases, such as rheumatoid arthritis, glomerulonephritis, atherosclerosis, and allograft rejection. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cytokine; Membrane protein, integral Chromosomal Location of Human Ortholog: 1p13. 3 Cellular Component: extracellular space; perinuclear region of cytoplasm Molecular Function:cytokine activity; growth factor activity; macrophage colony stimulating factor receptor binding; protein binding; protein homodimerization activity Biological Process: macrophage differentiation; osteoclast differentiation; positive regulation of cell migration; positive regulation of cell proliferation; positive regulation of cell-matrix adhesion; positive regulation of cellular protein metabolic process; positive regulation of macrophage differentiation; positive regulation of monocyte differentiation; positive regulation of mononuclear cell proliferation; positive regulation of osteoclast differentiation; positive regulation of protein kinase activity; reproductive developmental process; transmembrane receptor protein tyrosine kinase signaling pathway
NCBI Summary:	The protein encoded by this gene is a cytokine that controls the production, differentiation, and function of macrophages. The active form of the protein is found extracellularly as a disulfide-linked homodimer, and is thought to be produced by proteolytic cleavage of membrane-bound precursors. The encoded protein may be involved in development of the placenta. Alternate splicing results in multiple transcript variants. [provided by RefSeq, Sep 2011]
UniProt Code:	P09603
NCBI GenInfo Identifier:	311033367
NCBI Gene ID:	1435
NCBI Accession:	P09603. 2
UniProt Secondary Accession:	P09603,Q13130, Q14086, Q14806, Q5VVF3, Q5VVF4, Q9UQR8 A8K6J5,
UniProt Related Accession:	P09603
Molecular Weight:	29,215 Da
NCBI Full Name:	Macrophage colony-stimulating factor 1
NCBI Synonym Full Names:	colony stimulating factor 1
NCBI Official Symbol:	CSF1
NCBI Official Synonym Symbols:	MCSF; CSF-1
NCBI Protein Information:	macrophage colony-stimulating factor 1
UniProt Protein Name:	Macrophage colony-stimulating factor 1
UniProt Synonym Protein Names:	Lanimostim
Protein Family:	Macrophage colony-stimulating factor
UniProt Gene Name:	CSF1
UniProt Entry Name:	CSF1_HUMAN

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (pg/mL)	RLU	Average	Corrected
800	47435 57849	52642	52616
400	20295 23173	21734	21708
200	10515 8901	9708	9682
100	4450 4654	4552	4526
50	2341 2035	2188	2162
25	1141 979	1060	1034
12.50	507 511	509	483
0	26 26	26	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human MCSF were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human MCSF were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	37.28	71.87	375.90	39.45	68.71	338.75
Standard deviation	4.82	7.70	41.54	3.45	7.72	30.32
C V (%)	12.93	10.71	11.05	8.75	11.24	8.95

Recovery

The recovery of Human MCSF spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	91-107	98
EDTA plasma (n=5)	87-101	94
Cell culture media (n=5)	92-109	100

Linearity

Samples were spiked with high concentrations of Human MCSF and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	93-110	99-115	88-104
1:2	Average (%)	101	105	95
1:4	Range (%)	84-99	93-104	87-99
1:4	Average (%)	91	99	93
1:8	Range (%)	87-102	98-110	102-117
1:8	Average (%)	93	104	109
1:16	Range (%)	93-105	92-105	89-103
1:16	Average (%)	99	98	96

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.